1078 Preclinical report
`
`Superior antimetastatic effect of pemetrexed-Ioaded
`gelatinase-responsive nanoparticles in a mouse
`metastasis model
`Nannan Lua, Gin Liub, Rutian Lib, Li Xieb, Jie Shenb, Wenxian Guand
`,
`Xiaoping Gianb, Lixia Yub, Yitao Ding d
`, Xiqun Jiang C and Baorui Liua,b
`
`Novel pemetrexed-Ioaded gelatinase-responsive
`nanoparticles were prepared as a targeted delivery
`system to determine its potential for clinical therapy
`of malignant melanoma. The pemetrexed-Ioaded
`poly(ethylene glycol)(PEG)-peptide-poly(e-caprolactone)
`(PCl) nanoparticles included a gelatinase-cleavage
`peptide and a PEG-PCl-based structure. The
`pemetrexed-Ioaded PEG-peptide-PCl nanoparticles
`have shown the best anti metastatic effect in
`experimental lung metastasis models. The
`expressions of CD1 33 and thymidylate synthetase
`of metastatic tumors were also evaluated in our
`studies. Our results showed that pemetrexed-Ioaded
`gelatinase-responsive nanoparticles may represent
`a potent drug delivery system for inhibiting
`pulmonary metastasis and our preclinical results
`can provide new avenues for clinical therapy of
`
`malignant melanoma. Anti-Cancer Drugs 23:1078-1088 ©
`2012 Wolters Kluwer Health I Lippincott Williams & Wilkins.
`
`Anti-Cancer Drugs 2012, 23:1078-1088
`
`Keywords: anti metastasis, PEG-peptide-PCL nanoparticles, pemetrexed,
`targeting delivery system
`
`aThe Comprehensive Cancer Center of Drum Tower Hospital Affiliated to
`Nanjing Medical University, bThe Comprehensive Cancer Center of Drum Tower
`Hospital, Medical School of Nanjing University and Clinical Cancer Institute of
`Nanjing University, cLaboratory of Mesoscopic Chemistry and Department of
`Polymer Science and Engineering, College of Chemistry and Chemical
`Engineering, Nanjing University and dDepartment of General Surgery, Medical
`School of Nanjing University, Nanjing, People's Republic of China
`
`Correspondence to Baorui Liu, Comprehensive Cancer Center of Drum Tower
`Hospital Affiliated to Nanjing Medical University, Nanjing 210008, People's
`Republic of China
`Tel/fax: + 86 258 310 7081; e·mail: baoruiliu@nju.edu.cn
`
`Nannan Lu and Oin Liu contributed equally to the writing of this article.
`
`Received 12 December 2011 Revised 10 May 2012
`Revised form accepted 12 June 2012
`
`Introduction
`The targeting delivery system for anticancer therapy has
`shown considerable potential in the target-to-nontarget
`ratio, drug residence at the target site, and improved
`cellular uptake and intracellular stability. Active targeting
`plays an important role in the ultimate localization of
`therapeutic nanoparticles.
`
`Matrix metalloproteases (MMPs), a zinc-dependent family
`of endopeptidase, modulate cell-cell and cell-extracelluar
`matrix interactions and affect the cell phenotype by
`regulating the expression of E-cadherin and integrin [1].
`Most importantly, MMPs play a major role in cancer
`progression, including tumor growth, invasion, metastasis,
`and angiogenesis [2]. In particular, MMP2 and MMP9
`have been found in most tumor cells and to be involved in
`degradation of the extracellular matrix, cancer cell pro(cid:173)
`liferation and apoptosis, tumor angiogenesis and vasculo(cid:173)
`genesis, epithelial to mesenchymal transition, etc. [3].
`Nano-drug-targeted MMPs have been reported in some
`studies and the MMP-targeted drug delivery system has
`shown antitumor superiority [4-5].
`
`Pemetrexed disodium is a novel anticancer agent and has
`a wide range of applications in many cancers, including
`non-small-cell lung carcinomas and malignant mesothe(cid:173)
`liomas. Pemetrexed has also been reported for use in the
`treatment of other cancers in clinical trials, such as
`
`carcinomas of the head and neck, ovary, prostate, breast,
`and uterine cervix [6--12]. However, adverse effects have
`been reported, such as myelosuppression, hepatic en(cid:173)
`zyme elevations, maculopapular rash, emesis, fatigue,
`mucositis, and diarrhea [13]. Some studies have reported
`a correlation between the folic acid status of patients and
`the incidence of toxicity with pemetrexed [14,15]. A recent
`report of a clinical trial has indicated that toxicities are
`markedly decreased with folic acid supplementation in
`patients with advanced gastric cancer [16]. A preliminary
`comparative toxicity analysis of patients treated in different
`trials with or without vitamin supplementation has also
`shown that the addition of folic acid and vitamin B1z
`markedly reduced the incidence of adverse events, such as
`neutropenia, thrombocytopenia, and mucositis [17]. Some
`clinical studies have indicated that patients could benefit
`treatment combined with vitamin
`from pemetrexed
`supplementation [18,19]. Daily oral folic acid and vitamin
`B1z supplementation has been included as part of the
`treatment with pemetrexed of all patients undergoing
`the targeted drug delivery
`clinical
`therapy. However,
`system for pemetrexed has the potential to significantly
`improve the therapeutic outcome of cancer treatments
`while minimizing the side effects. There is no report on the
`use of therapeutic nanoparticles for
`the delivery of
`pemetrexed, and the use of a targeted drug delivery
`system for this is very important.
`
`0959·4973 © 2012 Wolters Kluwer Health I Lippincott Williams & Wilkins
`
`DOl: 10.1 097/CAD.Ob013e328356dc11
`
`Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1081-0001
`
`

`
`Both poly(ethylene glycol) (PEG) and polY(E-caprolactone)
`(PCL) are FDA-approved biodegradable and biocompatible
`materials and have been used widely in the biomedical
`field [20,21]. In our previous work, biodegradable amp hi(cid:173)
`philic copolymer mPEG-PCL and gelatinase-responsive
`copolymer mPEG-peptide-PCL were synthesized for the
`local delivery of docetaxel [22]. The nanoparticles con(cid:173)
`sisted of gelatinase-cleavable peptide and an mPEG-PCL(cid:173)
`based structure. Characterization of nanoparticles, in-vitro
`cellular uptake, real-time biodistribution of nanoparticles,
`and in-vivo antitumor efficacy of docetaxel-loaded nano(cid:173)
`particles were studied systemically [22]. Compared with
`mPEG-PCL nanoparticles without the gelatinase-cleavable
`peptide, the PEG-peptide-PCL nanoparticles showed
`higher intracellular uptake, higher local tumor accumulation
`and retention in the long run, and better in-vivo antitumor
`efficacy [22]. In this paper, we further evaluated the
`pharmacologic activity of the PEG-peptide-PCL nanopar(cid:173)
`ticles on lung metastasis of melanoma. We synthesized
`pemetrexed-loaded PEG-peptide-PCL nanoparticles. The
`characterizations, drug-loading content, and encapsulation
`efficiency, security and release of nanoparticles were
`studied in this paper. Especially, we determined the
`antimetastatic effect of nanoparticles and examined the
`expressions of CD133 and thymidylate synthetase (TS) of
`metastatic tumors in experimental lung metastasis models.
`
`Materials and methods
`Materials
`The pemetrexed disodium was provided by Eli Lilly
`Company (Indianapolis, Indiana, USA). Methoxy-poly(cid:173)
`ethyleneglycol-NHS was custom-made from Beijing
`Jiankai Technology Co. (Beijing, China). The gelati(cid:173)
`nase-cleavable peptide (PVGLIG) was custom-made
`from Shanghai HD Biosciences Co. (Shanghai, China).
`Gelatinases were purchased from Sigma-Aldrich (St
`Louis, Missouri, USA). E-Caprolactone (E-CL, Sigma(cid:173)
`Aldrich) and dimethyl formamide were purified by
`dehydration over CaHz at
`room
`temperature and
`extracted under reduced pressure. DMEM culture was
`obtained from Gibco (Grand Island, New York, USA).
`Cell Counting Kit-8 was obtained from Dojindo (Kuma(cid:173)
`moto, Japan). All other chemicals were of analytical grade
`and were used without further purification. Murine
`melanoma cell line B 16 was obtained from the Shanghai
`Institute of Cell Biology (Shanghai, China). Male C57/B6
`mice were purchased from the animal center of Drum
`Tower Hospital (Nanjing, China).
`
`Synthesis of nanoparticles
`Synthesis of PEG-peptide-PCL and PEG-PCL
`copolymers
`The PEG-peptide-PCL copolymers were synthesized as
`described previously [32]. The PEG-PCL copolymers
`were synthesized for pemetrexed-loaded PEG-PCL nano(cid:173)
`particles using the method previously described [23].
`
`Gelatinase-responsive nanoparticles Lu et a/. 1079
`
`Preparation of pemetrexed-/oaded nanopartic/es
`Pemetrexed-loaded nanoparticles were prepared according
`to a previously described method [23]. Briefly, a certain
`amount of vehicle and pemetrexed was dissolved in 1 ml of
`dichloromethane. The mixture was emulsified in 3 ml of a
`5% polyvinyl alcohol solution (w/v) by sonication (XL2000,
`Misonix, Farmingdale, New York, USA) for 30 s and was
`converted
`into an oil/water emulsion. The emulsion
`obtained was added to 8 ml of a 1 % polyvinyl alcohol
`solution (w/v) and subjected to sonication again, and then
`stirred to remove dichloromethane at room temperature in
`a fume cupboard. The resulting solution was filtered
`through a filter membrane to remove non incorporated
`drugs and copolymer aggregates. The nanoparticle solution
`was freeze dried with 3% mannitol and stored at 4°C. Drug(cid:173)
`free nanoparticles were produced in a similar manner, but
`without the addition of pemetrexed.
`
`Characterization of nanopartic/es
`The particle size and polydispersity of the pemetrexed(cid:173)
`loaded PEG-PCL nanoparticles and the pemetrexed(cid:173)
`loaded PEG-peptide-PCL nanoparticles were measured
`by dynamic light scattering (DLS; Brookhaven Instru(cid:173)
`ments Corporation, Holtsville, New York, USA). Particle
`size was the average of triplicate measurements for a
`single sample. The morphological characteristics of the
`nanoparticles were examined by transmission electron
`microscopy (TEM; JEM-lOOS; NEC Corporation, Tokyo,
`Japan). One drop of the pemetrexed-loaded PEG(cid:173)
`peptide-PCL nanoparticle suspension was placed on a
`copper grid overlaid with a nitrocellulose membrane and
`air-dried before observation.
`
`Drug-loading content and encapsulation efficiency
`A predetermined amount of freeze-dried nanoparticles
`was weighed and dissolved in the mobile phase. The
`drug-loading content and encapsulation efficiency were
`measured by high-performance liquid chromatography
`(C18 column; Agilent Technologies Ltd, Santa Clara,
`California, USA). The mobile phase consisted of
`0.01 mol/l NaHzP04 solution (pH 3.0) and methyl
`cyanides (82: 18, v/v); the column was eluted at a flow
`rate of 1.0 ml/min at room temperature with a wavelength
`of 227 nm for the detection of pemetrexed.
`
`The drug-loading content and encapsulation efficiency
`were calculated using the following equations:
`
`Drug-loading content (%)
`=Weight of the drug in nanoparticlesj
`weight of the nanoparticles xl 00 % ,
`
`Encapsulation efficiency ( % )
`=Weight of the drug in nanoparticlesj
`weight of the feeding drug x 100 % .
`
`(1)
`
`(2)
`
`Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1081-0002
`
`

`
`1080 Anti-Cancer Drugs 2012, Vol 23 No 10
`
`In-vitro release of pemetrexed-Ioaded nanopartic/es
`The lyophilized pemetrexed-loaded PEG-PCL nanopar(cid:173)
`ticles and pemetrexed-loaded PEG-peptide-PCL nano(cid:173)
`particles were suspended in PBS, pH 7.4, with or without
`1 mglml gelatinases and the pemetrexed concentration of
`the solutions was 100 flglml. The pemetrexed solution
`was placed in a dialysis bag with a 12 kDa molecular
`weight cutoff (Sigma-Aldrich). The dialysis bags were
`immersed into PBS at 3 TC. Periodically, the total release
`medium was withdrawn and replaced with fresh PBS,
`pH 7.4. The amount of pemetrexed released was
`measured by high-performance liquid chromatography.
`The experiments were repeated three times.
`
`Zymography
`B16 cells (1 x 107
`) were cultured in Dulbecco's modified
`Eagle medium (DMEM) medium without blood serum
`for 24 h and the culture solution was concentrated using
`centrifugal concentrators (Vivaspin, Sartorius, Goettin(cid:173)
`gen, Germany). The concentration of the loaded protein
`was 10 flg as determined using the BCA protein assay kit
`(Beyotime, Shanghai, China). Gelatinolytic activity was
`analyzed using the method described previously [24]. For
`zymography, a 5% spacer gel and 10% separation gel
`containing 0.1% gelatin (Sigma-Aldrich) were used. Mter
`electrophoresis, the gel was washed with washing buffer
`(2.5% TritonX-100) to remove the SDS for 1 h. Mter
`incubation with incubation buffer (20 mmol/l Tris-HCI
`(pH 8.0), 150 mmol/l NaCI, 5 mmol/l CaClz) overnight at
`3TC, the gels were stained with Coomassie blue R250 for
`4 h, and then destained with 30% methanol and glacial
`acetic acid. The destaining process was not interrupted
`until the clear band appeared.
`
`Cell viability assay
`B16 cells (5 x 10") were placed in a 96-well plate and
`cultured by DMEM medium at 3 TC. When the cells
`adhered, they were treated with several doses of free
`pemetrexed, pemetrexed-loaded PEG-PCL nanoparti(cid:173)
`cles, and pemetrexed-loaded PEG-peptide-PCL nano(cid:173)
`particles. After incubation for 48 h, 20 fll of the Cell
`Counting Kit-8 (Dojindo) was added to each well for an
`additional 2 h. The absorbance at 450 nm was read by a
`microplate reader (Bio-Rad, Hercules, California, USA).
`The entire experiment was repeated three times.
`
`Antimetastatic effect in experimental lung metastasis
`models
`The animal experiments were performed following the
`guidelines in the Guide for the Care and Use of Laboratory
`Animals published by the US National Institutes of Health
`(NIH publication No. 85-23, revised 1985), and was
`approved by the Animal Care Committee at Drum Tower
`Hospital, Nanjing, China.
`
`B16 cells (1 x 10'0) resuspended in 200 fll PBS were
`injected into the tail vein of six-week-old C57/B6 mouse
`
`and the day was designated as 'Day 0'. At day 11, mice
`were randomized
`into five groups and each group
`included five mice. The mice were treated intravenously
`with pemetrexed-loaded PEG-peptide-PCL nanoparti(cid:173)
`cles, pemetrexed-loaded PEG-PCL nanoparticles, free
`pemetrexed, empty nanoparticles, and saline on days 11,
`14,17, and 20, respectively. The pemetrexed solution was
`administered at a dose of 4 mglkg. The pemetrexed(cid:173)
`loaded nanoparticles were administered as a saline
`solution at the equivalent pemetrexed dose of 4 mglkg.
`The weight of each mouse was measured every day
`throughout the experiment. On day 21, the mice were
`euthanized and the lung tissues were collected for further
`study. After necropsy, lung metastasis was evaluated
`qualitatively by macroscopic photographs and quantita(cid:173)
`tively by determining lung weight [25].
`
`Hematoxylin and eosin staining
`The mice were killed and the tissue samples of the lung
`were instantly fixed
`in buffered formalin overnight,
`followed by dehydration with a tissue processor for 16 h.
`The tissues were embedded by paraffin. The 7 flm
`sections were prepared for H&E staining and microscopic
`examination.
`
`Immunohistochemistry
`Mter B16 tumor samples were fixed by formalin and
`embedded using embedding equipment, 4 flm sections
`were prepared. The sections were baked at 60°C for
`60 min, followed by
`lO-min washes with xylene. The
`rehydration of tissues was performed by 5-min washes
`in 100, 95, 85, and 70% ethanol and distilled water,
`respectively. Antigen retrieval was performed by heating
`the samples at 100°C for 20 min in 10 mmol/l sodium
`citrate (pH 6.0). Endogenous peroxidase activity of the
`tissue was blocked by incubation in 3% hydrogen peroxide
`in methyl alcohol for 15 min. The sections were incubated
`with rabbit anti-mouse polyclonal antibody (Wuhan Boster
`Biotechnology, Wuhan, China) at 3TC for 2 h. The sections
`the secondary
`were then incubated for 30 min with
`antibody (EliVision plus polyer HRP IHC Kit, Fuzhou
`Maixin Biotechnology, Fuzhou, China). Subsequently, the
`samples were redyed by hematoxylin. Dehydration was
`then performed following a standard procedure, and the
`slides were sealed with cover slips.
`
`RNA extraction and reverse transcription polymerase
`chain reaction analysis
`The RNA of metastatic tumor of lungs was extracted
`according to a previously described method [26]. The
`samples were
`resuspended
`in 300 fll RNA
`lysate
`[lOmmol/1 Tris-HCI (pH 8.0), 0.1 mmol/l EDTA (pH
`8.0), 2% SDS (pH 7.3), and 500 flglml proteinase K] for
`16 h at 60°C. The RNA was extracted by chloroform and
`phenol. The isopropanol and sodium acetate were used
`for precipitation at - 20°C for 1 h. The RNA was washed
`
`Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1081-0003
`
`

`
`Gelatinase-responsive nanoparticles lu et a/. 1081
`
`Table 1 Diameters and polydispersity of the nanoparticles
`
`Fig. 1
`
`Nanoparticles
`
`Diameter (nm)a
`
`Polydispersitya
`
`PEG-PCl
`PEG-peptide-PCl
`
`171.1 ±7.0
`231.3±5.3
`
`O.112±O.057
`O.187±O.139
`
`PCl, poly(c-caprolactone); PEG, poly(ethylene glycol).
`aThe SD value was for the mean particle size obtained from
`measurements.
`
`the three
`
`•
`
`with 70% ethanol and dissolved in 50 fll of RNase-free
`water. The RNA was reverse-transcribed to cDNA with
`reverse-transcriptase (Takara, Kyoto, Japan). Quantitative
`PCR was performed using the SYBR green qPCR kit
`(Takara) by a fluorescent temperature cycler (Mx3000P
`Real-Time PCR System, Stratagene; Agilent Technolo(cid:173)
`gies Inc.). The target cDNA sequences were amplified
`with the following primers: GAPDH 5'-AMTGGTGA
`AGGTCGGTGTG-3' and 5'-TGMGGGGTCGTTGATG
`G-3'; thymidylate synthase 5' -GGAGTAGACCAGCTGA
`AMGTG-3' and 5' -GATATGTGCMTCATGTAGGTGA
`G-3'. CDl33: 5'-ACCMCACCMGMCMGGC-3' and
`5' -GGAGCTGACTTGMTTGAGG-3'. The
`following
`PCR conditions were used: denaturation at 95°C for
`1 min, followed by 40 cycles of denaturation at 95°C for
`30 s, and annealing and extension at 60°C for 1 min. Each
`real-time PCR was repeated three times and the levels of
`mRNA expressions were calculated according to the
`comparative Ct method using GAPDH as an internal
`standard. The results were analyzed using the 2-MC,
`method [27].
`
`Statistical analysis
`Analysis was presented as means±SD. Differences within
`the
`treatment groups were analyzed by
`two-factor
`analysis of variance methods and the t-test. Differences
`were considered significant when P was less than 0.05.
`
`Results
`Characterization of the nanoparticles
`in an
`The size of pemetrexed-loaded nanoparticles
`aqueous solution was measured by DLS (Table 1). Figure 1
`the TEM micrographs of pemetrexed-loaded
`shows
`PEG-peptide-PCL nanoparticles. TEM images show
`that the nanoparticles were almost spheroid with a size
`of 200-250 nm each, which was consistent with the data
`from DLS (around 230nm).
`
`Drug-loading content and encapsulation efficiency
`Table 2 shows the encapsulation efficiency and the drug(cid:173)
`loading content of pemetrexed-loaded PEG-PCL nano(cid:173)
`particles under the different feeding ratios of copolymer
`and pemetrexed. With increased feeding, the encapsula(cid:173)
`tion efficiency and the drug-loading content increased.
`The P3 nanoparticles had the highest encapsulation
`efficiency and drug-loading content: 98 and 37.5%,
`respectively. The yield of P3 nanoparticles was higher
`than 95%, indicating no obvious loss of material during
`
`Transmission electron microscopy micrographs of PEG-peptide-PCL
`nanoparticles.
`
`the preparation process. The PEG-peptide-PCL nano(cid:173)
`particles were formed by the feeding ratio of P3, and the
`encapsulation efficiency and the drug-loading content
`were calculated to be 86.8 and 37.45%. In the following
`evaluation in vitro and in vivo, we used the pemetrexed(cid:173)
`loaded nanoparticles formed by the feeding ratio of P3
`because of its relatively higher encapsulation efficiency
`and drug loading.
`
`In-vitro release of pemetrexed-Ioaded nanoparticles
`Figure 2a shows the release profile of the pemetrexed(cid:173)
`loaded nanoparticles and both nanoparticles showed
`similar release profiles in the absence of gelatinases. An
`initial burst of more than 30% release in 3 h and a
`sustained release from these nanoparticles in the follow(cid:173)
`ing period were observed as shown in Fig. 2a. Actually,
`24 h of incubation with PBS caused approximately a total
`60% release. Figure 2b shows the release profile of the
`pemetrexed-loaded nanoparticles in gelatinases solution.
`The release of pemetrexed from pemetrexed-loaded
`PEG-peptide-PCL nanoparticles was faster than that of
`pemetrexed-loaded PEG-PCL nanoparticles in gelati(cid:173)
`nases solution (26.7 vs. 20% at 1 h, and 73.4 vs. 61.8%
`at 96 h). These results suggest that the gelatinases can ac(cid:173)
`celerate the release of pemetrexed from pemetrexed-loaded
`PEG-peptide-PCL nanoparticles, and
`the pemetrexed(cid:173)
`loaded nanoparticles may be used as a controlled-release
`system for the following in-vitro and in-vivo evaluations.
`
`Zymography
`the MMPs
`to measure
`Zymography was performed
`activities in melanoma cells. Figure 3a shows the results
`of zymography and indicates that melanoma cells secreted
`
`Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1081-0004
`
`

`
`1082 Anti-Cancer Drugs 2012, Vol 23 No 10
`
`Influence of drug feeding on the drug-loading content and
`Table 2
`encapsulation efficiency
`
`Feeding ratio
`(pemetrexed/copolymer)
`
`P1
`(2 mg/10 mg)
`
`P2
`(4mg/10mg)
`
`P3
`(6 mg/10 mg)
`
`OLC (%)
`EE (%)
`
`6.39
`22.7
`
`30.3
`80.9
`
`37.5
`98
`
`OLC, drug·loading content; EE, encapsulation efficiency.
`
`Fig. 2
`
`(a)
`
`100
`
`(b)
`
`MMp·2
`
`MMp·9
`
`MMp·2
`
`MMp·9
`
`Fig. 3
`- - - - - - - - - - - - - - - - - - - - - - - -
`(a)
`B16
`
`""O~
`~~
`OJ OJ
`.::. 0>
`OJ CIl
`
`80
`
`60
`
`40
`
`E c
`OJ OJ
`D..()
`OJ Q;
`> D..
`.~ "'0
`:; OJ
`(()
`E CIl
`:::J OJ
`O~ 20
`
`•
`PEG-PCl
`... • ... PEG-peptide-PCl
`
`0
`
`0
`
`(b)
`
`100
`
`20
`
`60
`40
`Release time (h)
`
`80
`
`100
`
`Detection of gelatinase (MMP2/9) expression (a) MMP2 and MMP9
`activities was carried out by zymography. (b) Immunohistochemical
`analysis of the gelatinases (MMP2/9) expression in B 16 tumors
`(x 400). MMP, matrix metalioprotease.
`
`""O~ 80
`~~
`OJ OJ
`.::. 0>
`OJ CIl
`
`60
`
`E c
`OJ OJ
`D..()
`OJ Q;
`> D..
`.~ "'0
`:; OJ
`(()
`E
`CIl
`:::J OJ
`O~
`
`---+---- PEG - PCl + gelatinase
`_____ PEG - peptide - PCl + gelatinase
`
`40
`
`20
`
`0
`
`0
`
`20
`
`60
`40
`Release time (h)
`
`80
`
`100
`
`The gelatinase·stimuli characterization of PEG-peptide-PCl
`nanoparticies. (a) Cumulative in·vitro release profile of pemetrexed from
`nanoparticies in the absence of gelatinases. Data are presented with
`SD. (b) In·vitro release of pemetrexed from nanoparticies in the
`presence of gelatinases. Data are shown with mean±SD. PCl,
`poly(£·caprolactone); PEG, poly(ethylene glycol).
`
`MMP2 and MMP9 proteins. The previous studies also
`obtained consistent results [24,28,29].
`
`Immunohistochemical analysis
`We observed MMP2/9 expression by immunohistochem(cid:173)
`ical analysis and MMP2/9 expression was considered to be
`positive when the cytoplasm of B16 cells turned brown.
`Our results showed that B16 cells expressed MMP2 and
`MMP9, and consistent results were obtained from
`zymography (Fig. 3b).
`
`In-vitro cytotoxicity
`Figure 4a shows the cytotoxicity of the free pemetrexed,
`two kinds of pemetrexed-loaded nanoparticles, and PBS.
`The treated groups showed similar concentration-depen(cid:173)
`dent cytotoxicity. There was no significant difference in
`cytotoxicity between free pemetrexed and the two kinds
`of pemetrexed-loaded nanoparticles.
`
`Inhibition of pulmonary metastasis after treatment with
`PEG-peptide-PCL nanoparticles
`Figure 4b shows the experimental design. B16 cells were
`administered by a tail vein injection to enable lung
`metastasis during a lO-day period. The metastasis burden
`refers to the relative increase in the total lung weight. Lung
`metastatic burden in mice treated with the pemetrexed(cid:173)
`loaded PEG-peptide-PCL nanoparticle group was the
`smallest among all the groups (Fig. 4c, the lung of a normal
`mouse expressed l.O±O.00095% of body weight). We did
`not observe apparent changes in body weight in any of the
`groups; no toxicity or adverse effects of empty nanoparti(cid:173)
`cles were observed during the entire treatment period
`(Fig. 4d). Macroscopic figures of lung metastasis from five
`treatment groups are shown and the black arrowheads
`indicate metastatic nodules (Fig. 5). Both free pemetrexed
`and the two kinds of pemetrexed-loaded nanoparticles
`effectively inhibited tumor metastasis. Pemetrexed-loaded
`PEG-peptide-PCL nanoparticles showed the best anti(cid:173)
`metastatic effect among all the treatment groups. The lung
`tissues in five groups were observed in the H&E-stained
`
`Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1081-0005
`
`

`
`Fig. 4
`
`(a) 120
`
`100
`
`80
`
`60
`
`~
`15
`CIl
`.;;
`~ 40
`
`Gelatinase-responsive nanoparticles lu et a/. 1083
`
`(b)
`
`B 16 tumor cells
`injection i.v.
`
`11-20
`
`21d
`
`o
`I
`
`20
`
`--+- Pemetrexed-Ioaded PEG-peptide-PCL nanoparticles
`____ Pemetrexed-Ioaded PEG-pel nanoparticles
`----..- Free pemetrexed
`__ PBS
`O~==~====~====~====~~~
`0.01
`0.1
`10
`100
`Equivalent pemetrexed concentration (Jlg/ml)
`
`4 X i.v. injections
`
`Every 3 days
`
`(Contain pemetrexed 4 mg/kg every group)
`N=5 C57/B6 mice/group
`
`• Saline
`o Pemetrexed-Ioaded PEG-peptide-PCL nanoparticles
`o Pemetrexed-Ioaded PEG-pel nanoparticles
`o Free pemetrexed
`o Empty nanoparticles
`
`(d)
`
`150
`
`"1J 100
`o
`~
`1:
`0>
`'w
`~ 50
`"1J o
`III
`
`(c)
`
`2.5
`
`2
`
`1.5
`
`1:
`0>
`'w :;:
`g> 0.5
`:::J
`--I
`
`o
`
`---t- Saline
`• , .• . , Empty nanoparticles
`-,t- • - Free pemetrexed
`-
`--+-- Pemetrexed-Ioaded PEG-PCl
`nanoparticles
`. . Pemetrexed-Ioaded PEG-peptide-PCl
`nanoparticles
`
`'1
`
`-
`
`11 12131415 16 17 18192021
`Days post cells challenge
`
`Cytotoxicity and anti metastatic effect of pemetrexed-Ioaded nanoparticies in vitro and in vivo. (a) Cytotoxicity of pemetrexed-Ioaded nanoparticles,
`free pemetrexed, and PBS against B16 cells for 48 h. Experiments were repeated three times and the results are shown as mean±SD from five
`samples in each group. (b) Experimental design of the experimental lung metastasis model. (c) lung metastasis burden on day 21 in the respective
`treatment groups. lung metastasis burden was calculated according to the formula: lung weight/body weight x 100%. Data are shown with
`mean±SD. (d) Body weight was monitored during the treatment period. Data are presented as mean±SD (n =5). i.v., intravenously; PCl,
`poly(£-caprolactone); PEG, poly(ethylene glycol).
`
`sections and the black arrowheads indicate metastatic
`tumor cells (Fig. 6).
`
`differences were observed in the expression levels of
`C0133 among the other three therapeutic groups (P> 0.05).
`
`The expressions of CD133 and thymidylate synthetase
`of metastatic tumors
`The TS expression level of metastatic tumors in lung
`tissues was shown in the experimental lung metastasis
`model (Fig. 7a). The group receiving pemetrexed-loaded
`PEG-peptide-PCL nanoparticles showed
`the highest
`expression of TS among the treatment groups. The
`expression level of TS in the free pemetrexed group was
`lower than that in the saline group. The C0133 expression
`of metastatic tumors in lung tissues was also analyzed in all
`the treatment groups (Fig. 7b). The C0133 expression of
`the group receiving PEG-peptide-PCL nanoparticles was
`the highest among all the groups. However, no significant
`
`Discussion
`In the present work, we prepared the PEG-peptide-PCL
`nanoparticles with a gelatinase-cleavable peptide. We
`systemically
`studied
`the characterization of PEG(cid:173)
`peptide-PCL nanoparticles and the antitumor superiority
`of docetaxel-loaded PEG-peptide-PCL nanoparticles [22]
`on the basis of particle size, morphology, and drug-loading
`content. In this study, we provided novel insights into the
`antimetastatic efficacy of PEG-peptide-PCL nanoparticles
`in a preclinical mouse model.
`
`Cytotoxicity tests showed no significant differences in
`cytotoxicity among these three therapeutic groups. A
`
`Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1081-0006
`
`

`
`1084 Anti-Cancer Drugs 2012, Vol 23 No 10
`
`Fig. 5
`
`(a)
`
`(b)
`
`(c)
`
`(d)
`
`(e)
`
`(f)
`
`Macroscopic pictures of lung metastasis from five treatment groups. (a) Normal lung tissue, (b) saline, (c) empty nanoparticles, (d) free pemetrexed,
`(e) pemetrexed-Ioaded PEG-PCl nanoparticles, and (f) pemetrexed-Ioaded PEG-peptide-PCl nanoparticles (n =5, the black arrowheads indicate
`metastatic nodules).
`
`Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1081-0007
`
`

`
`Fig. 6
`
`(a)
`
`(b)
`
`Gelatinase-responsive nanoparticles lu et a/. 1085
`
`(c)
`
`(d)
`
`(e)
`
`Representative H&E pictures of lung metastasis in an experimental lung metastasis model. (a) Saline, (b) empty nanoparticies, (c) free pemetrexed,
`(d) pemetrexed-Ioaded PEG-PCl nanoparticies, and (e) pemetrexed-Ioaded PEG-peptide-PCl nanoparticies (black arrows indicate metastatic
`tumor celis, x 40).
`
`reasonable explanation for this may be its sustained-release
`profile. The sustained release of the pemetrexed-loaded
`nanoparticles ensured that the concentration of peme(cid:173)
`trexed increased slowly in the culture medium [30--32].
`
`Despite substantial progress in oncotherapy, metastasis is
`still the primary cause of severe morbidity and mortality in
`cancer. The occurrence of metastasis in cancer patients is
`linked to a poor prognosis and remains a challenge for
`tumor treatment [33-34]. The pemetrexed-loaded gelati(cid:173)
`nase-responsive nanoparticles showed an antimetastatic
`effect and the inhibition of tumor metastasis was more
`significant than that of tumor growth only. We assessed the
`antimetastatic effect in an experimental lung metastasis
`model. Statistical analysis showed that the total tumor
`burden in the lung that received the PEG-peptide-PCL
`nanoparticles was significantly lower than that in the saline
`group (P < 0.05) as determined by a t-test. Thus, the
`PEG-peptide-PCL nanoparticles showed the best anti(cid:173)
`metastatic efficacy among the four therapeutic groups. The
`possible mechanism underlying the precedence of the
`PEG-peptide-PCL nanoparticles over
`the PEG-PCL
`
`nanoparticles can be deduced from the gelatinase-stimuli
`mechanism of the PEG-peptide-PCL nanoparticles. Many
`studies have shown that most of the tumor cells secreted
`MMP2 and MMP9 and our experiments also provided
`consistent results by measuring the MMPs activities of
`B16 cells (Fig. 3a and b) [24,28,29]. The gelatinases
`degraded the specific substrates, the linkers of hydrophilic
`PEG, and hydrophobic PCL blocks. The PEG-peptide
`conjugates were cleaved by gelatinases, which were
`specifically secreted by B16 cells in the tumor microenvir(cid:173)
`onment. The remaining PCL blocks gathered together for
`high hydrophobicity and had a higher affinity for tumor
`cells than PEGylated nanoparticles. The resulting PEG(cid:173)
`uncoated nanoparticles interacted efficiently with cancer
`cells, thus increasing the exposure of cancer cells to
`pemetrexed. The sustained release and the constant
`plasma concentration of pemetrexed might be responsible
`for the superiority of the pemetrexed-loaded PEG-PCL
`nanoparticles to the free pemetrexed.
`
`In previous studies, variations in body weight and the
`adverse effects of the PEG-peptide-PCL nanoparticles
`
`Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1081-0008
`
`

`
`1086 Anti-Cancer Drugs 2012, Vol 23 No 10
`
`Fig. 7
`
`(a)
`
`3.5
`
`3
`
`2
`
`c o
`'w ({)
`~
`D..
`><
`OJ
`OJ 2.5
`gJ
`CD
`..c
`C
`in'
`-¥J 1.5
`>,
`"1J
`'E
`>,
`£
`g;
`.~ 0.5
`&
`
`expression level of TS compared with that in the saline
`group, whereas the group treated with free pemetrexed
`showed lower TS expression than that in the saline group.
`The results showed that the metastatic tumors in the
`PEG-peptide-PCL nanoparticles group were more resis(cid:173)
`tant to pemetrexed.